Highly sensitive and quantitative detection of a minute amount of a detection object substance such as protein and DNA in laboratory tests makes it possible to perform treatment by quickly determining the patient's condition. There is therefore a need for a method for quantitatively measuring a minute amount of detection object substance with high sensitivity.
Surface plasmon-field enhanced fluorescence spectroscopy (hereinafter abbreviated as “SPFS”) is known as a method for detecting the detection object substance with high sensitivity. The SPFS utilizes a phenomenon in which surface plasmon resonance (hereinafter abbreviated as “SPR”) is caused when a metal film irradiated with light under a certain condition. A capturing body (for example, primary antibody) which can be specifically coupled with a detection object substance is immobilized on a metal film, and thus a reaction site for specifically capturing the detection object substance is formed. When a sample containing the detection object substance is provided to the reaction site, the detection object substance is coupled with the reaction site. Next, when anther capturing body (for example secondary antibody) labeled with fluorescence material is provided to the reaction site, the detection object substance coupled with the reaction site is labeled with the fluorescence material. When the metal film is irradiated with excitation light in that state, the fluorescence material which labels the detection object substance is excited by the electric field enhanced by SPR, thus emitting fluorescence. Accordingly, the presence or the amount of the detection object substance can be measured by detecting the fluorescence. In SPFS, a fluorescence material is excited by the electric field enhanced by SPR, and therefore the detection object substance can be detected with high sensitivity.
SPFS is roughly categorized by the way of coupling excitation light and surface plasmon, into prism coupling (PC)-SPFS and grid coupling (GC)-SPFS. In PC-SPFS, a prism in which a metal film is formed on one surface is used. In this method, excitation light is totally reflected at the interface between the prism and the metal film to couple excitation light and surface plasmon. PC-SPFS is the mainstream method in recent years; however, PC-SPFS is disadvantageous in terms of downsizing of the measurement device since a prism is used and the incident angle of excitation light to the metal film is large in PC-SPFS.
In contrast, in GC-SPFS, excitation light and surface plasmon are coupled together by utilizing diffraction grating (see PTL 1 and non-PTL 1). GC-SPFS can downsize the measurement device in comparison with PC-SPFS since a prism is not used and the incident angle of excitation light to the diffraction grating is small in GC-SPFS.